Yttrium oxide nanoparticles ameliorates calcium hydroxide and calcium titanate nanoparticles induced genomic DNA and mitochondrial damage, ROS generation and inflammation

Calcium hydroxide (Ca(OH)2NPs), calcium titanate (CaTiO3NPs) and yttrium oxide (Y2O3NPs) nanoparticles are prevalent in many industries, including food and medicine, but their small size raises concerns about potential cellular damage and genotoxic effects. However, there are very limited studies available on their genotoxic effects. Hence, this was done to investigate the effects of multiple administration of Ca(OH)2NPs, CaTiO3NPs or/and Y2O3NPs on genomic DNA stability, mitochondrial membrane potential integrity and inflammation induction in mouse brain tissues. Mice were orally administered Ca(OH)2NPs, CaTiO3NPs or/and Y2O3NPs at a dose level of 50 mg/kg b.w three times a week for 2 weeks. Genomic DNA integrity was studied using Comet assay and the level of reactive oxygen species (ROS) within brain cells was analyzed using 2,7 dichlorofluorescein diacetate dye. The expression level of Presenilin-1, tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) genes and the integrity of the mitochondrial membrane potential were also detected. Oral administration of Ca(OH)2NPs caused the highest damage to genomic DNA and mitochondrial membrane potential, less genomic DNA and mitochondrial damage was induced by CaTiO3NPs administration while administration of Y2O3NPs did not cause any remarkable change in the integrity of genomic DNA and mitochondrial membrane potential. Highest ROS generation and upregulation of presenilin-1, TNF-α and IL-6 genes were also observed within the brain cells of mice administrated Ca(OH)2NPs but Y2O3NPs administration almost caused no changes in ROS generation and genes expression compared to the negative control. Administration of CaTiO3NPs alone slightly increased ROS generation and the expression level of TNF-α and IL-6 genes. Moreover, no remarkable changes in the integrity of genomic DNA and mitochondrial DNA potential, ROS level and the expression level of presenilin-1, TNF-α and IL-6 genes were noticed after simultaneous coadministration of Y2O3NPs with Ca(OH)2NPs and CaTiO3NPs. Coadministration of Y2O3NPs with Ca(OH)2NPs and CaTiO3NPs mitigated Ca(OH)2NPs and CaTiO3NPs induced ROS generation, genomic DNA damage and inflammation along with restoring the integrity of mitochondrial membrane potential through Y2O3NPs scavenging free radicals ability. Therefore, further studies are recommended to study the possibility of using Y2O3NPs to alleviate Ca(OH)2NPs and CaTiO3NPs induced genotoxic effects.


Ethical approval
The design of this study was approved by the MSA University Research Ethics Committee.This study was reported according to ARRIVE guidelines and also Animal handling and experimentations were conducted in accordance with the Guidelines of the National Institutes of Health (NIH) regarding the care and use of animals for experimental procedures.

Determination of the nanoparticles' tested dose
An acute toxicity test was used to determine the appropriate utilized dose of the tested nanoparticles according to OECD standards 420 as follow: Twenty male mice were divided into four groups: an untreated control group and three treated groups, each with five mice.The three treated groups were orally given 2000 mg/kg of Ca(OH) 2 NPs, CaTiO 3 NPs or Y 2 O 3 NPs separately, while mice of the negative control group were orally given deionized distilled water.All mice of the four groups were monitored for any symptoms or morphological behavior of toxicity during the first 24 h of nanoparticles administration and up to 14 days of administration.Based on the mice's survival and the OECD standards 420 12,13 , the used dose of the tested nanoparticles was 2.5% of the safety tested dose determined from the OECD test.

Experimental design
In this study, 25 male mice were randomly separated into five groups with 5 mice each (Fig. S1).The first group served as the negative control, while the second group was taken 50 mg/kg of Ca(OH) 2 NPs through oral administration, the third group was given 50 mg/kg of CaTiO 3 NPs orally, the fourth group was given 50 mg/ kg of Y 2 O 3 NPs, and the fifth group was given a combination of the tested nanoparticles orally at a dose level of 50 mg/kg each, three times a week for two consecutive weeks.After 24 h of the last administration, all mice of the five groups were put to death by cervical dislocation, dissected and their brains were extracted, frozen, and preserved at − 80 °C for further analysis.

Detection of reactive oxygen species generation
The generation of reactive oxygen species (ROS) within brain cells was detected using 2,7 dichlorofluorescein diacetate (DCFH-DA) according to 14 .This compound can penetrate cells and react with ROS to produce the fluorescent chemical dichlorofluorescein (DCF).The procedure involved homogenizing 50 mg of brain tissue in Phosphate buffered saline (PBS), then rinsing it twice with PBS.50 μl of the cell suspension was mixed with 50 μl of DCFH-DA (20 mM) and left to incubate in the dark for 30 min.The mixture was then placed on slides and imaged under a fluorescent microscope (OLYMPUS CKX 41) at 20× magnification.

Estimation of DNA damage level
The level of DNA damage induction within brain cells was measured using the alkaline Comet test with a pH level higher than 13 15 .Slides were dipped in normal melting agarose (1%) and the samples were gently minced, mixed with low melting agarose (0.5%) and then placed on coated normal agarose slides.Sides were kept in the dark for 24 h at 4 °C in cold lysis buffer.After lysis, the slides were placed in staining jars containing alkaline electrophoresis buffer for 15 min, then electrophoresed for 30 min at 25 V and 300 mA in the same alkaline buffer.Slides were then neutralized, fixed with cold absolute ethanol and stained with ethidium bromide prior imaging.The slides were finally photographed under an epi-fluorescent microscope at 200× magnification and TriTek Comet ScoreTM Freeware v1.5 was used to assess the extent of DNA damage by measuring tail length, %DNA in tail, and tail moment.

Studying the mitochondrial membrane potential
The integrity of mitochondrial membrane potential was studied in brain tissues of the five groups using the method previously described by Zhang and his colleagues 16 .Briefly: a suspension of brain cells in PBS was combined with the fluorescent Rhodamine-123 dye and incubated in the dark for an hour at 37 °C.After incubation, the cells were washed twice with PBS.Then the fluorescence light emitted by Rhodamine-123 was captured and analyzed using an epifluorescence microscope at 200× magnification.

mRNA expression levels of presenilin-1 and inflammatory genes
For measuring the mRNA expression level of presenilin-1 gene and inflammatory tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) genes, quantitative RTPCR was conducted.The Gene JET RNA Purification Kit was used to extract RNA from frozen brain tissues in a 1.5 ml micro-centrifuge tube.About 30 mg of brain tissues were mixed with 300 µl of lysis buffer and β-mercaptoethanol and vortexed for 10 s, then 600 μl of diluted proteinase K was added, vortexed and incubated for 10 min at 25 °C.The tubes were centrifuged for 10 min at 12,000×g, and then the supernatant was transferred to a new RNase-free micro-centrifuge tube.450 μl of 100% ethanol was added; the lysate solution was filtered through a GeneJet RNA Purification column and washed with washing buffer.Finally, 100 μl of nuclease-free water was introduced to the column, centrifuged for 60 s, and the RNA was eluted into a new micro-centrifuge tube, which was stored at − 80 °C.The RNA was then reverse transcribed into complementary DNA (cDNA) using the Revert Aid First Strand cDNA Synthesis Kit.For amplification, 1 μl of the cDNA of each sample for each gene was mixed with 0.5 µl of each forward and reverse primers listed in Table 1 17,18 for three genes (Presenilin, TNF-α and IL-6) along with 6 µl of SYBER green master mix and 4 μl of nuclease-free water to make a total of 12 μl.The RTPCR reaction was then run with an initial denaturation of 95 °C for 15 min followed by 35 cycles of denaturation at 95 °C for 15 s and annealing at for 30 s, and extension at 72 °C for 1 min.Then final extension was done at 72 °C for 10 min.The expression of the three www.nature.com/scientificreports/studied genes was measured using β-actin gene as a housekeeping gene and the comparative ΔΔCt method was used for calculating the fold change in the gene expression.

Statistical analysis
The findings from this study were displayed as mean ± SD and evaluated using SPSS (version 20) at a significance level of < 0.05.One-way analysis of variance (ANOVA) was used to determine the impact of Y 2 O 3 NPs coadministration with Ca(OH) 2 NPs and CaTiO 3 NPs on induction of DNA damage and expression level of presenilin-1, TNF-α and IL-6 genes.Duncan's test was carried out to determine the similarities and differences between the control and four treated groups.

Characterization of nanoparticles
Characterization of Ca(OH) 2 NPs, CaTiO 3 NPs and Y 2 O 3 NPs in our previous studies using XRD analysis, DLS and TEM confirmed the purity of purchased nanopowders along with stability and well distribution of suspended nanoparticles in deionized distilled water.Moreover, TEM imaging revealed the spherical morphology of Ca(OH) 2 NPs, CaTiO 3 NPs and Y 2 O 3 NPs with an average particles' size of 59.82, 88.79 and 14.00 nm, respectively 7,10,11 .

Generation of intracellular ROS
Staining of brain cells with 2,7 DCFH-DA was highly informative and revealed that the highest generation of ROS was seen in the brain tissues of mice orally administered Ca(OH) 2 NPs alone compared to negative control and three treated groups (Groups III, IV and V) as depicted in Fig. 1.A slightly higher amount of ROS was observed in the brain tissue of mice orally ingested CaTiO 3 NPs alone compared to those noticed in the brain cells of negative control group.The last two groups administered Y 2 O 3 NPs alone or in combination with Ca(OH) 2 NPs and CaTiO 3 NPs exhibited the lowest amount of ROS generation in comparison to the other treated groups and almost identical to the ROS generated in the negative control brain cells, as displayed in Fig. 1.

Induction of DNA damage
Results of the Comet assay are shown in Table 2 and examples of the Comet nuclei scored with intact and damaged DNA are shown in Fig. 2. As depicted in Table 2, oral intake of Ca(OH) 2 NPs for six separate days over a 2-week period induced the highest statistical significant elevations in the DNA damage measured parameters: tail length, %DNA in tail, and tail moment compared to their values in the brain tissues of mice administered CaTiO 3 NPs or Y 2 O 3 NPs separately or together simultaneously with Ca(OH) 2 NPs (Table 2).Similarly, oral ingestion of CaTiO 3 NPs six times over a 2 weeks caused statistical significant increases in %DNA in tail and tail moment compared to the negative control values but remained significantly lower than the Ca(OH) 2 NPs administered group values (Table 2).On the other hand, oral administration of Y 2 O 3 NPs alone (Group IV) or simultaneously with Ca(OH) 2 NPs and CaTiO 3 NPs (Group V) did not cause any statistical changes in the tail length and tail moment compared to the negative control group values as displayed in Table 2.

Integrity of mitochondrial membrane potential
As illustrated in Fig. 3, oral administration of Ca(OH) 2 NPs alone caused a highest damage to the mitochondrial membrane potential as manifested by the remarkable decrease in the fluorescence intensity emitted by Rhodamine-123 stained brain cells compared to the negative control (Group I) and other three treated groups (Groups III, IV and V).Similarly, the oral administration of CaTiO 3 NPs led to a high decrease in the intensity of emitted fluorescent light compared to that emitted from the negative control brain cells, but still higher than that emitted from brain cells of mice administered Ca(OH) 2 NPs alone.
On the other hand, minimal damage to the mitochondrial membrane potential was noticed in the brain cells of mice orally administered Y 2 O 3 NPs alone (Group IV) or in combination with Ca(OH) 2 NPs and CaTiO 3 NPs (Group V) as slight decreases in the intensity of emitted light were observed in mice given Y 2 O 3 NPs alone (Group IV) and almost no changes were seen in mice orally given Y 2 O 3 NPs with Ca(OH) 2 NPs and CaTiO 3 NPs (Group V).

mRNA expression level
The quantitative RTPCR results are summarized in Fig. 4 and showed that the expression level of the three studied genes: presenilin-1, TNF-α, and IL-6 genes was statistically significantly elevated in the brain tissues of mice orally given Ca(OH) 2 NPs compared to their expression level in the negative control and other three groups  www.nature.com/scientificreports/as seen in Fig. 4. Meanwhile no remarkable changes were observed in the expression level of presenilin-1 gene after CaTiO 3 NPs administration compared to the negative control expression level (Fig. 4).On the contrary, the expression level of presenilin-1, TNF-α, and IL-genes did not change significantly and remained at the expression level of negative control after administration of Y 2 O 3 NPs alone (Group IV) or in simultaneously with Ca(OH) 2 NPs and CaTiO 3 NPs (Group V) as depicted in Fig. 4.

Discussion
The study of the cytotoxic and genotoxic effects of nanomaterials and nanoparticles is crucial in the field of biotechnology as nanotechnology advances.Extensive uses of Ca(OH) 2 NPs and CaTiO 3 NPs in various industrial, medical, food and consumer products increase the incidence of human exposure to these nanoparticles.However, limited data are available on the effect of Ca(OH) 2 NPs and CaTiO 3 NPs on the integrity of genomic and mitochondrial DNA in vivo along with the recently discovered free radicals scavenging activity of Y 2 O 3 NPs.Therefore, the current study was undertaken to estimate the impact of Y 2 O 3 NPs coadministration with Ca(OH) 2 NPs and CaTiO 3 NPs on the integrity of genomic DNA and mitochondrial membrane potential in the mice brain tissues.
In this study tracking ROS generation demonstrated the highest generation of ROS within the brain cells of mice orally given Ca(OH) 2 NPs (Group II) compared to those generated by CaTiO 3 NPs (Group III) or Y 2 O 3 NPs (Group IV) separately or in combination with Ca(OH) 2 NPs (Group V) as shown in Fig. 1.These results are in consistent with the recent detection of excessive ROS generation after single oral administration of Ca(OH) 2 NPs in various mice tissues: brain, bone marrow, liver, heart, spleen and lung 10,19 .Moreover, our finding of excessive ROS generation after administration of CaTiO 3 NPs manifested the in vivo induction of oxidative stress by CaTiO 3 NPs and supported the recent discovery of ROS generation and oxidative stress induction by CaTiO 3 NPs in breast cancer MCF-7 cell line 20 .
Extra-ROS generation can cause single-and double-strand DNA breaks, which is a lethal form of DNA damage 21,22 .According to Mills study 23 , double stranded-DNA breaks play a key role in the initiation of protooncogenes and the pathogenesis of cancer, suggesting that increased intracellular ROS production can cause cancer since intensive ROS generation disrupts the balance between oxidants and antioxidants and attacks proteins, lipids, carbohydrates and DNA inducing lipid peroxidation, protein damage and destruction, oxidative DNA damage and alterations in DNA bases.Indeed, the alkaline Comet assay is a very sensitive technique in detecting both single-and double-stranded DNA breaks 15 .Consequently, the highest incidence of DNA damage induction demonstrated by Comet assay in the brain tissues of mice given Ca(OH) 2 NPs alone compared to the other three treated groups (Table 2 and Fig. 2) can be attributed to the aforementioned highest ROS generation by Ca(OH) 2 NPs attacking DNA and cause breakages of both single and double DNA strands.Similarly, a recent study by Mohamed 10 , manifested the induction of DNA breakages in the brain tissues of mice given orally Ca(OH) 2 NPs through increased generation of intracellular ROS.
Similarly, the detected remarkable DNA damage induction by CaTiO 3 NPs administration through significant increases in the percentage of damaged DNA in the tail and tail moment could be attributed to the increased ROS generation noticed within brain cells of mice orally given CaTiO 3 NPs alone compared to those generated in the negative control brain cells.High ROS consequently attacks genomic DNA and increases the amount of damaged DNA.However, non remarkable changes observed in the tail length value after CaTiO 3 NPs administration compared to the negative control value revealed the large size of fragmented DNA causing slow migration in the mini gel 15,23 .
On the other hand, oral administration of Y 2 O 3 NPs alone caused non remarkable changes in the integrity of genomic DNA as demonstrated by the observable non-significant changes in the tail length and tail moment compared to negative control levels.Similarly, simultaneous coadministration of Y 2 O 3 NPs with Ca(OH) 2 NPs and CaTiO 3 NPs caused a marked decrease in the incidence of DNA damage induction noticed after administration of Ca(OH) 2 NPs or CaTiO 3 NPs separately and became non-statistically different from the negative control levels (Table 2).This remarkable reduction in the DNA damage induction probably due to the recently discovered antioxidant properties of Y 2 O 3 NPs; Y 2 O 3 NPs act as a direct antioxidant, controlling and neutralizing the generated harmful ROS 8,9,24 .Our findings of minimal ROS generation within brain cells of mice that were given Y 2 O 3 NPs alone or in combination with Ca(OH) 2 NPs and CaTiO 3 NPs further confirmed the antioxidant and free radicals scavenging capabilities of Y 2 O 3 NPs.Consistent with our findings, Y 2 O 3 NPs showed a potent dose dependent antioxidant and neuroprotective effect against oxidative stress and apoptosis induction [25][26][27][28] .
For more understanding of the impact of Y 2 O 3 NPs coadministration with Ca(OH) 2 NPs and CaTiO 3 NPs on the integrity of genomic DNA, the mRNA expression of Presenilin-1, TNF-α, and IL-6 genes were measured.Presenilin-1 is responsible for cleaving amyloid protein that causes Alzheimer's disease and is overexpressed in Alzheimer's patients [29][30][31] .Results of RTPCR showed that Y 2 O 3 NPs coadministration with Ca(OH) 2 NPs and CaTiO 3 NPs highly declined presenilin-1 overexpression (Fig. 4) noticed after oral administration of Ca(OH) 2 NPs alone thus protecting brain cells from Alzheimer's risk caused by administration of Ca(OH) 2 NPs alone previously reported by the study of Li and his colleagues 30 .Moreover, a marked decreases in the expression of presenilin-1 genes after oral administration of Y 2 O 3 NPs indicating the potentiality of Y 2 O 3 NPs in protecting the brain cells from Alzheimer's disease.Alzheimer's and other neurological diseases have been linked to elevated inflammatory cytokines expression and secretion.For example, the expression level of IL-6 and TNF-α genes is elevated in Alzheimer's disease [30][31][32] .Overexpression of inflammatory mediators including IL-6 and TNF-α genes also increases the expression level of Presenilin-1 and β-amyloid precursor protein genes causing aggregation and accumulation of β-amyloid peptides in the brain tissues and increasing the risk of Alzheimer's disease 31,33 .A marked overexpression of TNF-α and IL-6 genes observed in the brain tissue of mice orally exposed to Ca(OH) 2 NPs or CaTiO 3 NPs (Fig. 4), suggesting inflammation induction and immune stimulation 19,28 .However, no significant difference was found in the expression level of TNF-α and IL-6 genes after administration of Y 2 O 3 NPs alone revealing the antioxidant and protective properties of Y 2 O 3 NPs on brain cells.Therefore, our findings regarding the remarkable high decreases observed in the expression level IL-6 and TNF-α overexpressed genes by administration of Ca(OH) 2 NPs or CaTiO 3 NPs alone may explain the restored normal gene expression of presenilin-1 after Y 2 O 3 NPs coadministration with Ca(OH) 2 NPs and CaTiO 3 NPs (Fig. 4).
Variations in mitochondrial membrane potential can assess mitochondrial function and cell health using fluorescent dyes 34 .Mitochondria are critical to cell survival and any damage leads to cell death and disease.Therefore, the integrity of mitochondrial membrane potential has been studied in this study.Screening brain cells stained with Rhodamine-123 dye showed that administration of Ca(OH) 2 NPs caused a marked damage to mitochondria as manifested by the high reduction in the emitted fluorescent light (Fig. 3).This result supports findings of Mohamed study which demonstrated that Ca(OH) 2 NPs are genotoxic and can cause mitochondrial damage and even neurodegenerative diseases like Alzheimer's.Brain tissue exposed to CaTiO 3 NPs showed less damage, but still some harm to mitochondria 10 .Meanwhile, administration of Y 2 O 3 NPs alone or with Ca(OH) 2 NPs and CaTiO 3 NPs showed no remarkable decrease in emitted fluorescent light and appeared safe for mitochondria confirming the protective effect of Y 2 O 3 NPs (Fig. 3).

Conclusion
From above findings, it is concluded that administration of Ca(OH) 2 NPs alone induced the highest genomic DNA damage, ROS generation, disruption of mitochondrial membrane potential and inflammation, while administration of CaTiO 3 NPs alone had less toxic effects than Ca(OH) 2 NPs.Contrary, administration Y 2 O 3 NPs alone did not alter ROS generation, inflammatory genes expression and mitochondrial membrane potential.More interestingly, coadministration of Y 2 O 3 NPs with Ca(OH) 2 NPs and CaTiO 3 NPs alleviated the Ca(OH) 2 NPs and CaTiO 3 NPs induced genotoxicity, disruption of mitochondrial membrane potential and inflammation.However, single dose of nanoparticles, single tissue and limited techniques were used in this study, therefore further studies are necessary to fully understand the possibility of using Y 2 O 3 NPs to overcome Ca(OH) 2 NPs and CaTiO 3 NPs induced toxicity using different doses and more techniques in various organs.

Figure 2 .
Figure 2. Representative photomicrograph for the observed Comet intact and damaged nuclei in the brain tissues of negative control group and Ca(OH) 2 NPs, CaTiO 3 NPs or/and Y 3 O 2 NPs administered groups.(A) Intact nuclei (B) Damaged nuclei.

Figure 3 .
Figure 3. Integrity of mitochondrial membrane potential using Rhodamine dye within the brain cells of (a) Negative control group, (b) Ca(OH) 2 NPs administered group, (c) CaTiO 3 NPs administered group, (d) Y 3 O 2 NPs administered group and (e) group administered Ca(OH) 2 NPs, CaTiO 3 NPs and Y 3 O 2 NPs simultaneously.

Figure 4 .
Figure 4. Expression level of Presenilin-1, TNF-α and IL-6 genes in in the brain tissues of the negative control group and groups orally administered Ca(OH) 2 NPs, CaTiO 3 NPs or/and Y 2 O 3 NPs.Results are expressed as mean ± SD and were analyzed using one-way analysis of variance followed by Duncan's test to test the similarity between the control and three treated groups.Means with different superscript letters indicates statistical significant difference at p < 0.05 between the compared groups for the same gene.

Table 1 .
Sequences of the used primers in qRT-PCR.

Table 2 .
Tail length (px), %DNA in tail and tail moment in the brain tissues of the negative control group and groups orally administered Ca(OH) 2 NPs, CaTiO 3 NPs or/and Y 2 O 3 NPs.Results are expressed as mean ± SD. Results were analyzed using one-way analysis of variance followed by Duncan's test to test the similarity between the control and the four treated groups.Means with different superscript letters indicates statistical significant difference between the compared groups in the same column.Vol:.(1234567890)Scientific Reports | (2024) 14:13015 | https://doi.org/10.1038/s41598-024-62877-4